All Domains of Cry1A Toxins Insert into Insect Brush Border Membranes

Abstract

A critical step in understanding the mode of action of insecticidal crystal toxins from Bacillus thuringiensis is their partitioning into membranes and, in particular, the insertion of the toxin into insect brush border membranes. The Umbrella and Penknife models predict that only ␣-helix 5 of domain I along with adjacent helices ␣-4 or ␣-6 insert into the brush border membranes because of their hydrophobic nature. By employing fluorescent-labeled cysteine mutations, we observe that all three domains of the toxin insert into the insect membrane. Using proteinase K protection assays, steady state fluorescence quenching measurements, and blue shift analysis of acrylodanlabeled cysteine mutants, we show that regions beyond those proposed by the two models insert into the membrane. Based on our studies, the only extended region that does not partition into the membrane is that of ␣-helix 1. Bioassays and voltage clamping studies show that all mutations examined, except certain domain II mutations in loop 2 (e.g. F371C and G374C), which disrupt membrane partitioning, retain their ability to form ion channels and toxicity in Manduca sexta larvae. This study confirms our earlier hypothesis that insertion of crystal toxin does not occur as separate helices alone, but virtually the entire molecule inserts as one or more units of the whole molecule.Insecticidal crystal proteins produced by Bacillus thuringiensis are of great commercial potential in the field of agriculture and health (1) by targeting a wide spectrum of crop pests and vectors of human diseases. Cry1A toxins are active against lepidopteran insects, which include agricultural pests. The toxins are produced by the bacterium in the stationary phase as inactive crystal protoxins (1). Activation of the 130-kDa protoxin to a 65-kDa active toxin occurs in the alkaline environment of the lepidopteran midgut. Crystal structures of the active toxin show that the toxin has three domains that are conserved through all the crystal toxins (2-7). Domain I is an ␣-helical bundle made of seven antiparallel ␣-helices. Domain II is a globin-like, wedge-shaped prism made of antiparallel ␤-sheets ending in predominant ⍀-loops, and domain III is a lectin-like ␤-sandwich. The protease-activated form of the toxin binds to receptors on the surface of the insect brush border membrane. Several receptors implicated in binding to the toxin include cadherins (8, 9), alkaline phosphatase, and one or more forms of aminopeptidases (10, 11), glycolipids (12, 13), and glycoproteins (14). The receptor-bound toxin has been proposed to undergo conformational changes (15, 16) before or after inserting into the membrane to form ion channels.Studies focused on insertion of Cry1A toxins into the insect membrane have limited their studies to two ␣-helices of domain I of Cry1A toxins, ␣-helix 4 and 5 based on the theoretical Umbrella model of insertion proposed early in the 1980s (17). There has been little analysis of the other regions of the toxin. Several studies using nonspecific proteases to det...

Mutations in Domain I Interhelical Loops Affect the Rate of Pore Formation by the Bacillus thuringiensis Cry1Aa Toxin in Insect Midgut Brush Border Membrane Vesicles

“…In agreement with these findings, ␣4 was shown to line the lumen of the pores (42). On the other hand, convincing evidence supporting previous suggestions that most of the toxin molecule may become imbedded in the membrane (3,39,60) has recently been reported (44,45).Thus, several models have been proposed for the mechanism of toxin insertion and pore formation (4,9,28,32,39,44,52,56). Although these models differ in the identities of the toxin segments that are suggested to insert into the membrane, they all imply that the toxin undergoes conformational changes following binding to the membrane surface.…”

Differential Protection of Cry1Fa Toxin against Spodoptera frugiperda Larval Gut Proteases by Cadherin Orthologs Correlates with Increased Synergism

“…4B, lanes 4 to 6). To possibly clarify this issue, we used a protease K protection assay (2,29) to reveal membrane-inserted toxin monomers and oligomers. As shown in Fig.…”

Two disulfide mutants in domain I of <i>Bacillus thuringiensis</i> Cry3Aa δ-endotoxin increase stability with no effect on toxicity

“…However, we cannot rule out the possibility that buried disulfide bridges may not be exposed to the reducing environment of the midgut lumen. In this case, a plausible explanation is that the entire domain I inserts into membrane as previously proposed, either as a whole protein [7,[12][13][14][15][16][17] or as a member of an oligomeric complex [18,23,35,36]. This would also result in no difference in toxicity between the wild-type and disulfide proteins.…”

“…There is also evidence that other domains of the protein are able to insert into membrane [16], and that several mutations in the domain I (α-helices 3 and 4) affect oligomer formation [18][19][20]. Several disulfide-bridge mutations generated by protein engineering which are related to the membrane partitioning mechanisms of Cry toxins are reviewed in [21].…”

“…Both models involve separation of the α-helices from the α-helical bundle, followed by their insertion into the target membranes resulting in the formation of ion pores. Other studies have proposed that the whole domain I inserts into the protein, without α-helical separation [7,[12][13][14][15][16][17].…”